Magnetizable bead detector

ABSTRACT

A ferromagnetic thin-film based magnetic field detection system used for detecting the presence of selected molecular species. A magnetic field sensor supported on a substrate has a binding molecule layer positioned on a side thereof capable of selectively binding to the selected molecular species. The magnetic field sensor can be substantially covered by an electrical insulating layer having a recess therein adjacent to the sensor in which the binding molecule layer is provided. An electrical interconnection conductor can be supported on the substrate at least in part between the sensor and the substrate, and is electrically connected to the sensor. The magnetic field sensor can be provided in a bridge circuit, and can be formed by a number of interconnected individual sensors.

CROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] This application is a divisional application of application Ser.No. 10/799,429 filed Mar. 5, 2001 for “Magnetizable Bead Detector” byMark C. Tondra which is a continuation-in-part application ofapplication Ser. No. 09/687,791, filed Oct. 13, 2000, for “MagnetizableBead Detector” by Mark C. Tondra and John M. Anderson, which claimspriority from Provisional Application No. 60/159,185, filed Oct. 13,1999 for “Magnetoresistive Bead Assay”.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to the detection of magnetizablebeads and, more particularly, to detection of magnetizable beads inconnection with biological and chemical assays.

[0003] Among the biomolecular detection methods used to detect selectedmolecules in the presence of other kinds of molecules mixed therewithare binding assays which are based on use of certain binding moleculesto capture through specific chemical bondings the molecules selected fordetection. Such specific bondings include polynucleic acid bondings orhybridizations involving DNA and RNA, antibody to antigen bondings, andvarious ligand to various receptor bondings. The detection of theselected molecules may be of primary interest in its own right, but mayinstead be of primary interest in indicating the presence of some otheranalyte molecule, species or organism.

[0004] One arrangement for implementing such a detection scheme is toprovide a sensor in which the binding molecules are relatively stronglyattached to a solid substrate. An assay is begun by applying a samplesolution containing various kinds of molecules possibly including themolecules selected for detection to the sensor along with labelmolecules attached to label beads (or particles) also present in thesample solution or in a supplemental solution concurrently also applied.The binding molecules through specific bondings, or recognition events,capture the selected molecules or the label molecules attached to labelbeads, or both, and thereafter hold them at the corresponding capturesites, i.e. the sites of the binding molecules undergoing such abonding.

[0005] Label molecules on label beads are needed so that the occurrenceof a recognition event leads to some measurable signal to indicate thata selected molecule was found present. One kind of label bead for doingthis is a paramagnetic material bead having magnetizations that dependon externally applied magnetic fields. Application of such an externallyapplied field forcefully draws away any unbound label beads leaving thebound label beads at the capture sites while also magnetizing thosebound label beads. Magnetic field detectors at the capture sites mustsense the anomalies introduced into the externally applied field by thepresence of bound label beads to produce the desired signals indicatingthe number of, and possibly the location of, such bound label beads.From this information the number of selected molecules, and kindsthereof, in the sample solution can be determined.

[0006] Such label beads can range in magnetic material composition frompure ferromagnetic material (e.g. permalloy) to small percentages ofparamagnetic material encapsulated in plastic or ceramic spheres. Asindicated above, such label beads are typically coated with a chemicalor biological species that selectively binds to the selected moleculesin an analyte of interest including DNA, RNA, viruses, bacteria,microorganisms, proteins, etc. to define the assay function, or the kindof recognition events, to be associated with that bead.

[0007] However, the label beads must typically be very small, that is,on the order of a few to tens of nanometers (nm) up to maybe a hundredor so or even up to a few microns in some instances. As a result theanomalies in an externally applied field will be very small. Thus, thereis a desire for suitable magnetic field detectors for use with suchbeads.

BRIEF SUMMARY OF THE INVENTION

[0008] The present invention provides a ferromagnetic thin-film basedmagnetic field detection system used for detecting the presence ofselected molecular species. The system has a magnetic field sensorsupported on a substrate with a binding molecule layer positioned on aside thereof capable of selectively binding to the selected molecularspecies. The magnetic field sensor is substantially covered by anelectrical insulating layer which in some embodiments has a recesstherein adjacent to the sensor in which the binding molecule layer isprovided. An electrical interconnection conductor is supported on thesubstrate at least in part between the sensor and the substrate in someembodiments, and is electrically connected to the sensor. The magneticfield sensor can be provided in a bridge circuit, and can be formed by anumber of interconnected individual sensors located adjacent to thebinding molecule layer. A polymeric channel and reservoir structure baseis provided for the system.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1 shows a coordinate definition schematic diagram,

[0010]FIG. 2 shows a magnetizable sphere based magnetic response fieldschematic over a structure,

[0011]FIG. 3 shows a direction lines schematic for a magnetizable spherein a uniform externally applied field,

[0012]FIG. 4 shows field magnitude value bands for the field of FIG. 3at a selected plane,

[0013]FIG. 5 shows an alternative magnetizable sphere based magneticresponse field schematic over a structure,

[0014]FIG. 6 shows a detector device portion embodying the presentinvention,

[0015]FIG. 7 shows an electrical schematic diagram providing arepresentation of FIG. 6,

[0016]FIGS. 8 and 9 show layer diagrams of a detector device portionembodying the present invention,

[0017]FIG. 10 shows a detector device portion embodying the presentinvention,

[0018]FIG. 11 shows a layer diagram of a detector device portionembodying the present invention,

[0019]FIG. 12 shows a graph having a plot representing performance of acomponent used with the present invention,

[0020]FIGS. 13, 14 and 15 show detector device portions embodying thepresent invention,

[0021]FIGS. 16 and 17 show layer diagrams of a detector device portionembodying the present invention,

[0022]FIGS. 18, 19 and 20 show detector device portions embodying thepresent invention,

[0023]FIGS. 21 and 22 show layer diagrams of detector devices portionembodying the present invention,

[0024]FIG. 23A shows a layer diagram of a detector device portion andFIG. 23B shows a top view of that device portion both embodying thepresent invention,

[0025]FIG. 24 shows a detector device portion embodying the presentinvention,

[0026]FIGS. 25, 26 and 27 show layer diagrams of detector devicesportion embodying the present invention,

[0027]FIG. 28 shows a detector device portion embodying the presentinvention,

[0028]FIG. 29 shows a layer diagram of the detector device portion ofFIG. 28,

[0029]FIG. 30 shows a part of the detector device portion of FIGS. 28and 29, and

[0030]FIG. 31 shows a characteristic diagram of a detector device.

DETAILED DESCRIPTION

[0031] The need for a very sensitive magnetic field detector followsfrom the smallness of the label beads and that the field anomaliescaused thereby in an otherwise more or less uniform externally appliedmagnetic field are very localized in roughly decaying as 1/r³, where ris the magnitude of the distance from the center of a bead. Because ofthis highly localized field, only the sensor structure within about onebead diameter sees an appreciable magnetic field effect. Yet, of course,the largest signal possible from a given label bead is desired withoutsuffering nonlinear effects due to bead to bead interactions. Theapplied field disruption from a label bead that is detected by a sensoris proportional to the magnetization of that bead.

[0032] Commercially available paramagnetic beads have either a small, orare entirely devoid of, remnant magnetization (i.e., the magnetizationremaining in the bead after an externally applied magnetic field isremoved). Consequently, a magnetic detector arrangement requires anadditional externally applied magnetic field to magnetize the beads usedin any assay. The detector design must be such that detection can takeplace in an externally applied magnetic field optimal for detection, butnot have the detection sensor response saturated, and not mask the fieldanomaly provided by the presence of a label bead.

[0033] In these circumstances, the nature of a label bead anomalyinduced in an externally applied field can be approximated byconsidering a sphere, 10, of paramagnetic material present in a uniform,externally applied, magnetic field of magnitude H_(app) directed fromleft to right along the x axis in FIG. 1 parallel to the surfacesupporting the bead and a magnetic field detector, 11, showndiagrammatically therebelow. (Detector 11 is assumed here to notsignificantly affect the magnetic fields present.) Using mks units,magnetic induction B (Webers/meter²), magnetic field strength H, andmaterial magnetization M are related as (bold type denoting vectorquantities)

B=μ₀(H+M),

and

M=λ_(m)H_(app),

[0034] where λ_(m) is the dimensionless magnetic susceptibility. Theentities “magnetizable beads” is defined in terms of the susceptibilityλ_(m). While λ_(m) can be measured for any material, only some materialshave values substantially different from zero (λ_(m) of empty space iszero). A paramagnetic material has a single-valued λ_(m) just greaterthan zero, typically ˜0.001, while a ferromagnetic material has amultivalued λ_(m) (due to hysteresis) that is much larger than zero(typically ˜1). As will become clear in the following analysis, largervalues for λ_(m) lead to larger signals at the detector. However,ferromagnetic particles have a tendency to agglomerate because ofmagnetic attraction to one another (which potentially ruins an assay)and the multivalued nature of λ_(m) for such material leads todifficulties in quantification of surface coverage. Consequently, thetypical assay employs a paramagnetic material magnetizable bead with aslarge a λ_(m) as possible without causing agglomeration. A sphericalbead of a paramagnetic or ferromagnetic material will be uniformlymagnetized in a uniform externally applied magnetic field. The externalfield contribution from the magnetizing of bead 10 will have a dipoledistribution based on an effective dipole moment p of

p=M(4π/3)a ³

[0035] where a is the bead radius, and M (Webers/meter) is the beadmagnetization as indicated above.

[0036] If the origin is taken at the center of bead 10 in a sphericalcoordinate system as shown in FIG. 1, this dipole field will have noazimuthal angular dependence and can be represented by just two vectorcomponents. These two components of the dipole field at distance r fromthe center of bead 10 are

H _(r)=λ_(m) H _(app)(8π/3)_(a ³ /r ³)cos(θ),

and

H _(θ) =H _(app)(4π/3)_(a ³ /r ³)sin(θ)

[0037] where θ is the angle between r and M (which follows the directionof the externally applied field H_(app) along the x axis). The 1/r³dependence of these field components can clearly be seen.

[0038] The magnetic field contribution alone from bead 10 positionedover magnetic field detector structure 11 in a uniform applied fielddirected from left to right is roughly depicted in FIG. 2. Similarstructures and features in subsequent figures have the same numericaldesignations as were used in the preceding figures. The total fieldoutside this bead in such an externally applied field will be the sum ofthe uniform applied field directed from left to right and the fieldcontribution from bead 10 which contribution thus results in an anomalyin the otherwise uniform field present. Note that the field contributiondirectly under bead 10 in FIG. 2 (field contribution at θ=π/2 in FIG. 1)due to its magnetization will directly oppose the externally appliedfield thereby decreasing the total field value in this vicinity. Thetotal field H_(total) at magnetic field detector structure 11 directlyunder bead 10 is, as stated, the sum of the externally applied fieldH_(app) and the field contribution from bead 10 due to itsmagnetization, H_(B), or

H _(total) =H _(app) +H _(B) =H _(app) +H _(θ)

[0039] since the radial component does not contribute at θ=π/2.

[0040] Substituting for H_(θ) from above yields $\begin{matrix}{H_{total} = {H_{app}\left\{ {1 - \left\lbrack {{\chi_{m}\left( {4{\pi/3}} \right)}\left( {a^{3}/r^{3}} \right){\sin (\theta)}} \right\rbrack} \right\}}} \\{= {H_{app}{\left\{ {1 - \left\lbrack {{\chi_{m}\left( {4{\pi/3}} \right)}\_ \left( {a^{3}/r^{3}} \right)} \right\rbrack} \right\}.}}}\end{matrix}$

[0041] A plot of the direction lines of this total field about bead 10(independent of the azimuthal angle) is shown in FIG. 3 (which assumesthat flux arising because of permeable material in a detector structuretherebelow is negligible or is confined in a closed flux path therein).If the distance from the bottom surface of bead 10 to the upper surfaceof magnetic field detector structure 11 is equal to the radius of bead10 (i.e., at the point having coordinates r=2a, θ=π/2) then

H _(total) =H _(app)[1=λ_(m)(π/6)].

[0042] A practical superparamagnetic bead ( having a magnetization in amagnetic field much greater than ordinary paramagnetic material) has avalue for λ_(m) around 0.05, so the total field at magnetic fielddetector structure 11 would be on the order of

H _(total) =H _(app)[1−0.05(π/6)]≈(0.97)H _(app).

[0043] The effect of bead 10 is thus to provide an anomaly in theexternally applied field that has the effect of attenuating the totalapplied field at magnetic field detector structure 11 by about 3%, achange in field value from the value of the externally applied fieldwithin the range of detectability of the sensors to be described below.An alternative example provides a filler representation of this fieldattenuation situation as shown in FIG. 4 which shows the componentparallel to H_(app) of the field from the particle in the plane ofmagnetic field detector structure 11, this example based on using valuesof λ_(m)=0.05, a particle radius of 0.5 μm, and a particle-to-sensorseparation of 0.1 μm. But this attenuation effect is limited to theregion in the immediate vicinity of bead 10. The magnitude of thedistortion to the externally applied field because of the presence ofbead 10 drops off approximately as 1/r³ in separating from that bead inall directions so that bead 10 must be sufficiently close to magneticfield detector structure 11 if field attenuation in this range is tooccur at that structure. Hence, if a single bead is to be detected, itis best to match the size of magnetic field detector structure 11 to thesize of bed 10 for maximum resolution.

[0044] A suitable arrangement for magnetic field detector structure 11can be advantageously fabricated using ferromagnetic thin-film materialsbased on magnetoresistive sensing of magnetic states, or magneticconditions, thereabout. Such devices may be provided on a surface of amonolithic integrated circuit to provide convenient electricalinterconnections between the device and the operating circuitry thereforin the integrated circuit chip.

[0045] In the recent past, reducing the thicknesses of the ferromagneticthin-films and the intermediate layers in extended “sandwich” structuresin which the two major surfaces of the intermediate layer each havethereon an anisotropic ferromagnetic thin-film layer, including thosehaving additional alternating ones of such films and layers, i.e.superlattices, have been shown to lead to a “giant magnetoresistive(GMR) effect” being present. This effect yields a magnetoresistiveresponse which can be in the range of an order of magnitude or moregreater than that due to the well-known anisotropic magnetoresistiveresponse.

[0046] In the ordinary anisotropic magnetoresistive response, varyingdifferences between the direction of the magnetization vector in theferromagnetic film and the direction of the sensing current passedthrough the film lead to varying differences in the effective electricalresistance in the direction of the current. The maximum electricalresistance occurs when the magnetization vector in the film and thecurrent direction are parallel to one another, while the minimumresistance occurs when they are perpendicular to one another. The totalelectrical resistance in such a magnetoresistive ferromagnetic film canbe shown to be given by a constant value, representing the minimumresistance, plus an additional value depending on the angle between thecurrent direction in the film and the magnetization vector therein. Thisadditional resistance follows a square of the cosine of that angle.

[0047] As a result, operating external magnetic fields can be used tovary the angle of the magnetization vector in such a film portion withrespect to the easy axis of that film portion which comes about becauseof an anisotropy therein typically resulting from depositing the film inthe presence of a fabrication external magnetic field oriented in theplane of the film along the direction desired for the easy axis in theresulting film. During subsequent operation of the device with theresulting film, such operating external magnetic fields can vary theangle to such an extent as to cause switching of the film magnetizationvector between two stable states which occur as magnetizations orientedin opposite directions along that easy axis. The state of themagnetization vector in such a film portion can be measured, or sensed,by the change in resistance encountered by current directed through thisfilm portion.

[0048] In contrast to this arrangement, the resistance in the plane of aferromagnetic thin-film is isotropic with respect to the giantmagnetoresistive effect, rather than depending on the direction of asensing current therethrough as for the anisotropic magnetoresistiveeffect, depends on the cosine of the angle between magnetizations in thetwo ferromagnetic thin-films on either side of an intermediate layer. Inthe giant magnetoresistive effect, the electrical resistance through the“sandwich” or superlattice is lower if the magnetizations in the twoseparated ferromagnetic thin-films are parallel than it is if thesemagnetizations are antiparallel, i.e. directed in opposing directions.Further, the also present anisotropic magnetoresistive effect in verythin-films is considerably reduced from the bulk values therefor inthicker films due to surface scattering, whereas very thin-films are afundamental requirement to obtain a significant giant magnetoresistiveeffect.

[0049] In addition, as indicated, the giant magnetoresistive effect canbe increased by adding further alternate intermediate and ferromagneticthin-film layers to extend the “sandwich” or superlattice structure. Thegiant magnetoresistive effect is sometimes called the “spin valveeffect” in view of the explanation that a larger fraction of conductionelectrons are allowed to move more freely from one ferromagneticthin-film layer to another if the magnetizations in these layers areparallel than if they are antiparallel with the result that themagnetization states of the layers act as sort of a valve.

[0050] These magnetizations results often come about because of magneticexchange coupling between the ferromagnetic thin-films separated by theintermediate layers, these intermediate layers typically formed from anonferromagnetic transition metal. The effect of the exchange couplingbetween the ferromagnetic thin-film layers is determined to asubstantial degree by the thickness of such an intermediate layertherebetween. The effect of the coupling between the separatedferromagnetic thin-film layers has been found to oscillate as a functionof this separation thickness between these layers in being ferromagneticcoupling (such that the magnetizations of the separated layers areparallel to one another) and antiferromagnetic coupling (such that themagnetizations of the separated layers are opposed to one another, orantiparallel to one another). Thus, for some separation thicknesses, thelayer coupling can be of zero value between extremes of suchoscillations.

[0051] Exhibiting the giant magnetoresistive effect in a superlatticestructure, or in an abbreviated superlattice structure formed by a threelayer “sandwich” structure, requires that there be arrangements inconnection therewith that permit the establishment alternatively of bothparallel and antiparallel orientations of the magnetizations in thealternate ferromagnetic thin-film layers therein. One such arrangementis to have the separated ferromagnetic thin-films in the multilayerstructure be antiferromagnetically coupled but to a sufficiently smalldegree so that the coupling field can be overcome by an externalmagnetic field.

[0052] Another arrangement is to form the ferromagnetic thin-film layerswith alternating high and low coercivity materials so that themagnetization of the low coercivity material layers can be reversedwithout reversing the magnetizations of the others. A furtheralternative arrangement is to provide “soft” ferromagnetic thin-filmsand exchange couple every other one of them with an adjacentmagnetically hard layer (forming a ferromagnetic thin-film double layer)so that the ferromagnetic double layer will be relatively unaffected byexternally applied magnetic fields even though the magnetizations of theother ferromagnetic thin-film layers will be subject to being controlledby such an external field.

[0053] One further alternative arrangement, related to the first, is toprovide such a multilayer structure that is, however, etched into stripssuch that demagnetizing effects and currents in such a strip can be usedto orient the magnetizations antiparallel, and so that externallyapplied magnetic fields can orient the magnetizations parallel. Thus,parallel and antiparallel magnetizations can be established in theferromagnetic thin-films of the structure as desired in a particularuse. Such a structure must be fabricated so that any ferromagnetic orantiferromagnetic coupling between separated ferromagnetic films is nottoo strong so as to prevent such establishments of film magnetizationsusing practical interconnection arrangements.

[0054] A magnetic field sensor suited for fabrication with dimensions ofa few microns or less can be fabricated that provides a suitableresponse to the presence of very small external magnetic fields and lowpower dissipation by substituting an electrical insulator for aconductor in the nonmagnetic layer. This sensor can be fabricated usingferromagnetic thin-film materials of similar or different kinds in eachof the outer magnetic films provided in a “sandwich” structure on eitherside of an intermediate nonmagnetic layer which ferromagnetic films maybe composite films, but this insulating intermediate nonmagnetic layerpermits electrical current to effectively pass therethrough basedprimarily on a quantum electrodynamic effect “tunneling” current.

[0055] This “tunneling” current has a magnitude dependence on the anglebetween the magnetization vectors in each of the ferromagnetic layers oneither side of the intermediate layer due to the transmission barrierprovided by this intermediate layer depending on the degree of matchingof the spin polarizations of the electrons tunneling therethrough withthe spin polarizations of the conduction electrons in the ferromagneticlayers, the latter being set by the layer magnetization directions toprovide a “magnetic valve effect”. Such an effect results in aneffective resistance, or conductance, characterizing this intermediatelayer with respect to the “tunneling” current therethrough.

[0056] There are many types of magnetoresistive material that can beused. A sandwich type (e.g. 35A NiFeCo/15 CoFe/Cu 35/15 CoFe/35 NiFeCo)or a pinned sandwich (e.g. 35A NiFeCo/15 CoFe/Cu 35/40 CoFe/100 IrMn,also called a spin valve) are useful arrangements. They have lowersaturating fields the structures described for use below, and so aremore sensitive, but also more likely to be saturated by any beadmagnetizing fields. Anisotropic Magnetoresistive (AMR) films can also bepatterned into such devices. The key for all of these will be to get thebeads close to the sensitive layer or layers, and to magnetically biasthe magnetoresistors such that they give maximum sensitivity to thepresence of the beads. Another enhancement to the sensing material is togive it “hard edges” by oxidizing the edges, pinning them with someantiferromagnetic material, or constructing them so that the twomagnetic layers contact each other at the edge of the resistor stripe.Resistors prepared this way will have lower hysteresis in the range ofmagnetic field magnitudes relevant for the assay.

[0057] The GMR magnetoresistors typically used here are formed of asuccession of thin, alternating magnetic and nonmagnetic metallic layershaving an electrical resistance that is a function of an externalmagnetic field. This resistance, as stated above, varies with the angleθ between the magnetizations of adjacent magnetic layers with a sin θdependance, and a typical change from a minimum (i.e., magnetic layersare aligned in parallel) to maximum (i.e., when magnetic layers arealigned antiparallel) resistance is ˜10% of the minimum value. The thinsuccession of layers is fashioned into sensors as long, thin,rectangular structures but with pointed ends. The observed resistance isthen proportional to the length and inversely proportional to the width.A typical sheet resistance is 10 ohms/□. Consequently, such a typicalmagnetoresistor with a respective width and length of 0.5 and 5.0microns (10 squares) would have resistance of 100 ohms under parallelmagnetic layer magnetizations that increases to 110 ohms (10%) whenunder antiparallel magnetic layer magnetizations.

[0058] The preceding last example, however, assumes instead forconvenience that a 1 μm×1 μm GMR detector is positioned directly beneathparamagnetic label bead 10 so that this bead is situated above the uppersurface of structure 11 in the z direction. Such a detector wouldtypically have a current therethrough of 5 mA, and a 10 Ω to 11 Ωresistance change corresponding to H_(app) ranging from 200 Oe to 0 Oe.This represents the 10% resistance change over 200 Oe as indicated inFIG. J. The detector voltage, then, changes at a rate of 25 μV/Oe. Themaximum H_(app) possible without saturating the sensor is 200 Oe. Thefield attenuation from the bead modeled above is, on average, 0.05 timesthe H_(app). Though the total resistance change will be slightly lessthan the average field change due to current redistribution within theGMR sensor, assume for now that the net resistance change is exactlyproportional to the average field change. Hence, the maximum totalsignal field is 10 Oe (0.05×200 Oe). The voltage “signal” from the beadis then 250 μV.

[0059] The noise of the detector has two main components: thermal noiseand current dependent 1/f noise. The thermal (Johnson) noise for a 10 Ωresistor is 0.4 nV/{square root}{square root over (Hz)}. The detector'sintrinsic 1/f noise will typically be two orders of magnitude higherthan the thermal noise at 1 Hz, and have a corner frequency at 10 kHz.Assuming a 1 Hz measurement frequency, the total noise will be dominatedby the 1/f noise, and be about 40 nV/{square root}{square root over(Hz)}. So the signal to noise ratio at 1 Hz with a 1 Hz bandwidth is 250μV/40 nV=6250:1. While this simple calculation ignores engineeringchallenges that must be addressed to fully use the available signal, itis clear that detection of a single 1 μm diameter bead with a 1 μm×1 μmGMR detector is not limited by fundamental signal to noise issues.

[0060] The relative positional arrangement in FIG. 2 of bead 10 and GMRmagnetic field detector structure 11, and the relative orientation ofthe externally applied magnetic field, can be varied. One variation ofthe externally applied magnetic field relative orientation is to applysuch a field normal to the plane of GMR detector structure 11 as shownin FIG. 5. GMR detector structure 11 is about 1000 times more difficultto magnetize in the direction normal to its major surface than in adirection parallel thereto, and so a much larger magnetizing field canbe applied to bead 10 and that structure without saturating the GMRmagnetoresistors therein. Here, magnetic field detector structure 11,rather being shown by itself with bead 10 directly thereover, is shownover a space between that structure and another GMR magnetic fielddetector structure, 11.

[0061] The use of multiple magnetoresistors in GMR magnetic fielddetector 11 permits measurement circuit advantages including use of adifferential detection, e.g. a bridge circuit detector, arrangement.Although a bead detection site can be provided with only onemagnetoresistor as shown in FIG. 2, a bridge circuit having two or moremagnetoresistors therein offers several benefits including rejection ofcommon mode noise, and provision of first order temperaturecompensation. In such a bridge circuit, one or more of several GMRmagnetoresistors provided therein are exposed to possible magnetic fieldanomalies due the possible presence of bead 10 while the remainingmagnetoresistors are not so exposed, or are instead exposed to fields inthe opposite direction.

[0062] One such example is shown in FIGS. 6 and 7. A single beaddetection site, 12, formed by the presence of a binding moleculecoating, is provided having four GMR magnetoresistors, 13, 14, 15 and16, as individual sensors electrically connected together the metalinterconnections shown in a Wheatstone bridge arrangement between metalinterconnections voltage supply terminals, 17 and 18, and locatedbetween two flux concentrators, 19 and 20, to be described below. Site10 is provided so that one pair (active) of magnetoresistors, that is,13 and 14, are in opposite legs of the bridge circuit and subject to thefield anomaly introduced by the presence of bead 10 while the other pair(reference) formed of magnetoresistors 15 and 16 is not so subjectedeven they are also subjected to the externally applied field. The signaldeveloped across the bridge circuit is provided between two metalinterconnections signal terminals, 21 and 22, and provided to the signalinputs of a differential amplifier, 23, providing a correspondingdifferential output signal at its output, 24.

[0063] Keeping these two pairs of magnetoresistors distinguishable asactive magnetic field anomaly sensing magnetoresistors and referencemagnetic field sensing magnetoresistors during an assay can beaccomplished using one or another of several techniques. The simplestway is to provide a covering of a suitable kind such that no bead everreaches a position sufficiently close to the reference magnetoresistorsto magnetically affect them. Alternatively, the detection site can befabricated, or subsequently modified, so that few or no particles willable to adhere to a surface of, or a surface near, the referencemagnetoresistors. In addition, or instead, a passivation layer over thedetector surface (the upper or final layer shown over all else) can bekept thick over the reference magnetoresistors as in the layer diagramof FIG. 8 to substantially separate bead 10 therefrom but kept thin overthe active magnetoresistors (the upper or final two layers removed overstructure 11 with or without binding molecule coating 12 provided there)as shown in the layer diagram of FIG. 9 to allow bead 10 to approachclosely.

[0064] Flux concentrators 19 and 20 in the form of permeable materialmasses can be incorporated into a bridge circuit detection arrangementto concentrate externally applied magnetic fields in and about theactive magnetoresistors, as shown in FIGS. 6 and 9, or a fluxconcentrator, 25, can be provided to increase and guide magnetic fieldsdeveloped in the detector itself through providing current through aconductor coil, 26, formed thereabout as shown in FIG. 10. In eachinstance, both the active pair and the reference pair ofmagnetoresistors are shown positioned in the gap between two suchconcentrating flux concentrators, or in the gap between the two ends ofan enhancing and guiding flux concentrator, so that they all will beimmersed in substantially the same concentrated externally appliedmagnetic field.

[0065] In such an arrangement, the flux concentrator or concentratorsare simply acting as a multiplier of the externally applied magneticfield. The multiplication factor is approximately [concentratorlength]/[concentrator separation gap]. Alternatively, the referencemagnetoresistors can be positioned underneath the flux concentrators.They will in such a position experience a much smaller net field (theyare substantially shielded from the applied field) than the activemagnetoresistors positioned in the concentrator gap (the common modefield rejection is then lost) but they will require no space to beprovided for them in the gap.

[0066] The methods used to fabricated structures like those shown inFIGS. 8 and 9 are based on well known semiconductor device fabricationprocesses. Two alternative processes will be described, the choice ofwhich depends on whether the resulting devices are to haveinterconnection metal provided above or below the GMR magnetic fielddetector structure. If the interconnection metal is to be provided belowthe GMR magnetic field detector structure, a Si based wafer, 30, whichmay have monolithic integrated circuitry therein, has a 200 nm layer ofsubstrate Si₃N₄, 31, supported thereon, on which a 1.5 μm layer of Al isfirst deposited. This Al layer is patterned using standardphotolithography and a reactive ion etch (RIE) to form what will becomethe interconnections, 32, for the magnetic field detector, or assay,circuit. A 2.5 μm (must be at least as thick as the previous Al layer)thick layer of Si₃N₄ is deposited over the patterned Al. Then, theresulting surface is planarized using chemical mechanical polishing(CMP). The Al interconnections are exposed during the CMP, so that theresulting surface of the wafer has both Al and a Si₃N₄ base, 33, in it.Both materials are smooth. The GMR magnetic field detector structurematerial is then deposited.

[0067] The succession of alternating magnetic and nonmagnetic metallicthin-film layers in the GMR magnetic field detector structure is formedin a vacuum deposition system using rf diode sputtering. The successionthickness is 10 nm to 40 nm, depending upon the particular structurechosen for use. The thicknesses of individual layers within thesuccession are controlled to tenths of nanometers. The GMR succession isfollowed by providing thereon a 10 nm CrSi etch stop layer on which isfurther provided a 40 nm Si₃N₄ mask layer. The Si₃N₄ mask layer ispatterned using standard photolithography and an RIE etch. The GMRmagnetic field detector structure+etch stop succession is then etchedthrough the resulting Si₃N₄ mask using an ion mill to leave the GMRmagnetic field detector structure 11 magnetoresistors, 34. Most or allof the Si₃N₄ mask is etched away in the ion mill, and at least some orall of the CrSi layer, 35, below this mask remains on thesemagnetoresistors. At this point, the GMR magnetic field detectorstructure 11 magnetoresistors and Al interconnections are complete andcan be tested.

[0068] The GMR magnetoresistors and the Al interconnections are thenpassivated if desired by providing thereover a thin layer of Si₃N₄ as apassivation layer, 36. The required thickness of this passivation layerdepends upon two things, the step height of the GMR magnetoresistorsabove the base layer therebelow, and the standoff voltage requiredbetween the GMR elements and the bead samples that will be present abovethe passivation. If some passivation is required and the voltagerequirements are minimal, though non-zero (<10 Volts), the passivationlayer minimum thickness limitation will be the first requirement fortopological coverage of the GMR step edge. A rule of thumb is to use apassivation layer thickness equal to that of the step being covered,although less is possible. A passivation layer thickness of 10 nm istypically sufficient. Such a passivation layer provides a nominalstandoff voltage of about 1000 Volts/μm, so a 10 nm passivation willnominally provide 10 V. The resulting detector is shown in FIG. 11 withbinding molecule coating 12 provided thereon when desired over GMRmagnetic field detector structure 11 magnetoresistors 34.

[0069] If the interconnection metal is to be provided above the GMRmagnetic field detector structure, on the other hand, the structures ofthe layer diagrams in either of FIGS. 8 and 9 can be fabricated. In thissituation, the succession of alternating magnetic and nonmagneticmetallic thin-film layers in the GMR magnetic field detector structureis deposited directly upon substrate Si₃N₄ layer 31 on Si wafer 30 asabove. An 10 nm CrSi etch stop layer plus a Si₃N₄ masking layer of 40 nmthickness is deposited, and then photolithography and RIE are used topattern the Si₃N₄ to form an etching mask, followed again by use of anion mill to etch the GMR magnetic field detector structure+etch stopsuccession thereby leaving the GMR magnetic field detector structure 11magnetoresistors. Again, most or all of the Si₃N₄ mask is gone afterthis milling, and most or all of CrSi etch stop layer 35 under this maskremains on these magnetoresistors.

[0070] Thin Si₃N₄ passivation layer 36 is then deposited, serving hereas an inner passivation layer, and passageways, or vias, to the GMRmagnetic field detector structure 11 magnetoresistors are etched ininner passivation layer 36 using RIE. A relatively thick Al (˜1 μm)layer is next deposited and patterned using photolithography and RIE, sothat interconnections, 37, are made for the GMR magnetic field detectorstructure 11 magnetoresistors. The Al needs to be thick so that the leadresistance is low compared to the resistance of these magnetoresistors.Exposed surfaces of aluminum interconnections 37 and Si₃N₄ passivationlayer 36 then have deposited thereon a 10 mn aluminum nitride (AlN)layer, 38, for its adhesion properties followed by a 1.5 μm Si₃N₄ layer,39, which together form the outer passivation for the device. FIG. 8shows a layer diagram of the resulting structure at this point of thefabrication process which leaves an outer passivation layer so thickthat bead 10 thereon over GMR magnetic field detector structure 11magnetoresistors would have little magnetic effect on those referencemagnetoresistors or relatively less magnetic effect on actively sensingmagnetoresistors.

[0071] However, to provide actively sensing magnetoresistors also,photolithography and RIE are used to remove the Si₃N₄ portion of theouter passivation layer from selected areas or some portion of thatlayer thereover. Such areas include those over bonding pads for externalwiring and, of course, those over certain magnetoresistor sites whereenhanced sensitivity to magnetic particles is desired. Material millingin an ion mill step is used to remove the exposed AlN layer in theseregions in a controlled way, leaving nearly the full thickness of theunderlying inner Si₃N₄ passivation layer, or somewhat less if desired.FIG. 9 shows the layer diagram of the result upon the completion ofthese steps in the fabrication process if binding molecule coating 12and flux concentrators 19 and 20 are ignored.

[0072] Flux concentrators 19 and 20 can be added at this point foramplifying externally applied fields, or coil 26 and flux concentrator25 can be added at this point for guiding on-chip fields. A photoresistplating mold is formed, and permalloy is electroplated into the mold toa thickness of about 15 μm to form flux concentrators 19 and 20. FIG. 9shows the structure resulting to this point in the fabrication processif binding molecule coating 12 is again ignored.

[0073] There are many methods to provide binding molecule coating 12,i.e. of preparing the assay probe region, the particulars of whichdepend upon the intended purpose of the assay. One method to createprobes for a DNA hybridization assay is described in the following.

[0074] The resulting structure after the flux concentrators arecompleted is coated with gold using a lift-off mask that leaves goldover large portions of the chip, but such that it does not shortdifferent magnetoresistor interconnections together. Then spots ofreagents containing thiolated DNA oligomers are dispensed over theintended assay probe region using a Sapphire-tipped Rapidograph pen. Inorder to keep the non-probe regions inert, the chip surface is alsotreated with thiolated PEG (O-(2-mercaptoethyl)-o′-methyl-polyethyleneglycol 5000. Such a material can be used to complete the device shown inFIG. 9 in providing coating 12 shown there.

[0075] The response of a typical GMR magnetic field detector structuremagnetoresistor to an external magnetic field is unipolar, linear, andsaturates in fields ranging from tens of Oe to a few hundreds of Oe,depending upon the particular magnetic design. A plot of the observedresistance vs. externally applied magnetic field strength for a typicalmultilayer GMR magnetic field detector structure magnetoresistor isshown in FIG. 12. The presence of a bead 10 is then detected byobserving a change in the resistance due to the external fieldexperienced by the magnetoresistor.

[0076] Detecting a single magnetizable bead with GMR magnetic fielddetector structure magnetoresistors at a binding molecule coated assaysurface, as essentially set out above, is typically insufficient in viewof the overall assay signal-to-noise ratio being proportional to thenumber of capture sites at the assay surface, i.e. proportional to thesquare root of the assay surface area over which such sites areprovided, assuming a random a real distribution of “noise events”. Assay“noise events” include situations like beads sticking where they are notsupposed to be sticking in the detection scheme (non assay-specificadhesion, leading to artificially high magnetic signal) and other kindsof particles (other than beads) sticking to detection sites in place ofbeads (contaminants, for instance, leading to artificially low magneticsignal).

[0077] However, individual assay surface sizes on the order of 10 to1000 μm in diameter often result if formed by techniques that are notlithographically based such as forming the assay surface sites by“spotting” the chip with fluid using a fine plotter-type device or atiny metal point dipped in an appropriate solution to provide thebinding molecule coating. These techniques result in spot sizes muchlarger than 1 μm. Lithographic techniques, instead, can result in muchsmaller individual effective detectors over the assay surface.

[0078] Thus, assay surfaces that are much larger than a typical bead,say a 50 μm sided square, lead to a desire to measure the surfaceconcentration of beads across the entire surface, or detection site, asaccurately as possible. This implies having a detector arrangement withan active region covering as much of the site as possible, and whichprovides a response to beads captured at the site that is uniform acrossthe site. A GMR magnetic field detector structure magnetoresistorarrangement having multiple effective individual detectors can beprovided across such a assay surface, or detection site, by havingseveral individual GMR magnetic field detector structuremagnetoresistors electrically connected together in series.

[0079]FIG. 13 shows a top view of such a detector arrangement in aportion of a device formed on an integrated circuit under a squarebinding molecule coating forming detection site 12 in which theindividual GMR magnetic field detector structure magnetoresistors areeach approximately 4 μm wide, separated by 2 μm, and connected togetherusing Al metal interconnections 37 formed over the top of the seriesconnected magnetoresistors. Passivation layers and any fluxconcentrators used are omitted from this view for clarity. Thesemagnetoresistors are individual sensors like the magnetoresistors inFIG. 6 and so designated 13, 13+, 13″, 13′″, 13″″, 13 ^(v), 13 ^(vi) and13 ^(vii) in representing an active group of magnetoresistors. The“active region”, or detection site 12, is a square 50 μm on a side thatis effectively defined by the existence of the binding molecule coating“assay surface” and possibly other construction features such as thinnedsensor passivation, etc.

[0080] In situations where a full Wheatstone bridge circuit is desiredto be used, the same assay surface can be monitored by two seriesconnected, interleaved GMR magnetic field detector structuremagnetoresistor groups connected as shown in FIG. 14 for again a portionof a detector device formed on an integrated circuit with some layersomitted for clarity. These groups are again taken as like themagnetoresistors in FIG. 6 and so designated 13, 13′, 13″ and 13′″ forone group of active resistors, and designated 14, 14′, 14″ and 14′″ forthe other group of active resistors. The gap between two adjacentmagnetoresistors is ideally as small as possible to maximize surfacecoverage, but is limited in coverage by practical lithographical spacingconsiderations. The width of each of the magnetoresistors can vary overa wide range, from the narrowest that can be fabricated (<0.5 μm) in thebest resolution fabrication process to as wide as the entire assay siteif only one or two magnetoresistors are to be used. The length-to-widthratio of each of the magnetoresistors is to be such that the resistancetotal of the groups satisfies detection circuit criteria. For example,some preamplifiers operate best with a source impedance of about 1 kΩwhich requires 100 squares of 10 Ω/square material. The resistor in FIG.13 has 8 legs, each 4 μm×50 μm=12.5 squares=125 Ω for a total of 1 kΩ.

[0081] The width of an individual magnetoresistor is also importantbecause the most sensitive region of such a magnetoresistor is in theinterior thereof because the edges thereof are magnetically relativelystiff in resistor rotation of the magnetization in those regions. Widerresistors, in so having a proportionally larger sensitive region, willthus be more sensitive. On the other hand, wider resistors require morecurrent to generate the same voltage signal because of reducedresistance. Too much current can generate excess heat which may disturbor destroy the coating forming the assay surface or affect the test inother ways.

[0082] The system sensitivity to a single bead is enhanced by usingmultiple GMR magnetic field detector structure magnetoresistors withrespect to such a bead so that more than one of those magnetoresistorsis affected by the magnetic field anomaly introduced by the presence ofa bead. Thus, rather than having the bead come to a position directlyabove a magnetoresistor, the bead would instead be directed to aposition between two adjacent magnetoresistors, or even in the areabetween four such magnetoresistors. A channel can be provided betweeneach member of a pair of magnetoresistors, or a hole could be providedin the area between four magnetoresistors, in which the beads would bemore likely to be captured, and in which the binding molecule coatingforming the assay surface would be placed. Such an arrangement is shownin the top view in FIG. 15 in which a bridge circuit is shown formed ina portion of the detector arrangement provided on a monolithicintegrated circuit having detection site 12 located between activemagnetoresistors 13 and 14 where each will experience the magnetic fieldanomaly introduced by the presence of a bead 10 bound to site 12 whilemore remotely located reference magnetoresistors 15 and 16 will not.

[0083] The depth of these channels or holes, 40, should be such that thecenter of the bead is level with the magnetoresistors in the verticaldirection as is shown in FIG. 16 which is a portion of a layer diagramof the device portion shown in FIG. 15 and an enhancement of thestructure shown in FIG. 9. This arrangement is advantageous when usingan externally applied excitation magnetic field that is directedparallel to the major surfaces of the magnetoresistors as in FIG. 15which could be alternating direction fields. The response magnetic fieldfrom the bead would be similarly directed, and the resulting poles ofthe bead would be very close to the magnetoresistor edge for properlychosen magnetoresistor dimensions. The alternative situation for thearrangement of FIG. 15 is to rely just on the binding molecule coatingto keep a bead 10 between magnetoresistors 13 and 14 as shown in FIG.17.

[0084] Word lines, or structures like them, can be used to generate suchexternally applied fields on-chip that either supplement or eliminateoff-chip magnetic field generating. Permeable mass flux keepers can beused to direct the flux from the lines and from the beads. Use of aburied word line would allow the beads to be close to themagnetoresistors as does the use of buried connectors as shown in FIG.11. Alternatively, the externally applied excitation magnetic fieldscould be directed perpendicularly to the major surfaces of themagnetoresistors but then off-chip magnetic field generating is likelyto be needed.

[0085] The FIG. 17 arrangement relying just on the binding moleculecoating to keep a bead 10 in place is ideal if patterning the bindingmolecule coating is performed to provide portions thereof just where themagnetizable beads are desired to be attached. This way, the beads willstick only where they impart the largest field to the magnetoresistors.The form of the pattern depends on what sensing mode is being used. Ifthe externally applied excitation magnetic field orientation isperpendicular to major surfaces of the magnetoresistors, the beadsshould come to be positioned next to or on top of the magnetoresistorsas shown in the top views of FIGS. 18 and 19 which alternatively showthe magnetoresistors interconnections above and below themagnetoresistors, respectively, in the shown detector portions. Sinceonly one magnetoresistor 13 is actively affected by bead 10 here, theother magnetoresistor in the bridge circuit previously described as alsobeing active in other arrangements has been redesignated 14′ here. Ifthe excitation orientation is parallel, the beads should come to bepositioned on the magnetoresistors or between two magnetoresistors as inFIGS. 15, 16 and 17. Of course, the binding molecule coating material towhich the beads come to stick in a position must be thin with respect tothe thickness of detection devices layers (<1 μm). If the coating isinstead tens of microns thick, then any magnetoresistor arrangement willbe relatively insensitive.

[0086] The magnetic field anomaly arising from the presence of a micronsized magnetizable bead is, in actuality, often very nonuniform and canbe best detected in a differential mode using a gradiometer magneticfield measurement arrangement. In most situations, this means a bridgecircuit configuration where one or two magnetoresistors of a fourmagnetoresistor bridge circuit experience the effects of the presence ofa bead while the other two are not. That is, the “active ”magnetoresistors are under the influence of the presence of a bead(since magnetoresistor 16 rather than magnetoresistor 13 is activelyaffected by bead 10 here along with magnetoresistor 14, thismagnetoresistor in the bridge circuit previously described as beinginactive in other arrangements has been redesignated 16′ here) and the“reference” magnetoresistors are not so influenced as in FIG. 20.

[0087] If embedded externally applied magnetic field excitation coilstraps and thick permeable masses as flux “pipes” are used on-chip togenerate such externally applied magnetic fields locally aroundmagnetoresistors, the presence of beads near the straps or resistorswill change the local field distribution. Such bead presence will alsochange the field distribution in the gap between two flux concentrators.If a magnetoresistor is in the gap between two flux concentrators, themagnetic field experienced by them in the gap for the occurrence of anexternally applied field will be reduced by the presence of a bead inthe gap because they concentrate flux therethrough from the gap regionand the magnetoresistors. A bridge sensor can be made to use thisproperty by putting two magnetoresistors in a gap between fluxconcentrators where beads are allowed, or even promoted, to stick whileputting two identical magnetoresistors in an otherwise identical gapbetween flux concentrators where beads are not allowed or promoted tostick. The externally applied magnetic field could be generated byon-chip current conductors located about the flux concentrators as inFIG. 10.

[0088] Beads near current conductors will redistribute magnetic fluxgenerated by the current as well. If the beads are on the same side ofthe conductors as a magnetoresistor as in FIG. 21, the current generatedmagnetic field at the magnetoresistor due to current in such aconductor, 41, will be reduced. If the beads are on the opposite side ofthe conductor from the magnetoresistor, such as a conductor, 41′, shownin FIG. 22, the current generated magnetic field will be increased. Inthe former situation, the beads form a higher magnetic permeability pathand concentrate flux away from the magnetoresistor. In the latter case,the lower permeability of the beads permits a larger amount of flux togo around the conductor for a given amount of current therethrough.

[0089] If a magnetoresistor is fabricated with the sensitive axisperpendicular to its direction of longest extent, with a buried,parallel current conductor provided around it at a fixed separation, thefield along the sensitive axis would be zero at the magnetoresistor. Ifa bead came to be positioned above the conductor on one side of themagnetoresistor, such as a conductor, 41″, shown in FIGS. 23A and 23B,the field would become unbalanced at the GMR resistor and a net fieldwould be experienced to provide the basis for a four-magnetoresistorbridge circuit to generate an offset dependent output signal.

[0090] Typical magnetizable beads for assays require magnetic fields ofseveral hundred to over a thousand Oe for saturation. Consequently,there is a mismatch between the field ranges of GMR magnetic fielddetector structure magnetoresistors and the fields required tosignificantly magnetize the beads. Because the saturation field for thebeads is so large compared to the magnetoresistors, an arrangement toprovide different flux densities for an externally applied excitationmagnetic field would help. This can be done by making a permeable massbased flux loop on the detector surface with two types of gaps thereinas shown in the detector arrangement portion shown in FIG. 24 with thepermeable masses designated 19′ and 20′. One gap is for beads in bindingmolecule coating region 12, and the other gap is for the activemagnetoresistors. The reference magnetoresistors are positioned underthe flux loop mass (shown schematically), or under other permeable massshields. Both gaps have the same length along sensitive axes of themagnetoresistors (though this is not necessary). The gap for beads wouldbe much narrower than the gap for the magnetoresistors. So while thesame flux would pass across both gaps, the flux density would be muchlower at the active magnetoresistor gap than at the bead gap. The fluxloop permeable mass would ideally have coil 26 wound therearound forcurrent to generate the externally applied field. Alternatively, thatfield could be generated by an underlying planar coil.

[0091] Sample solution to be applied in assays using the magneticdetectors in integrated circuits of the kinds described above,containing various kinds of molecules possibly including the moleculesselected for detection by the sensor along with label molecules attachedto label beads (or particles) also present in the sample solution or ina supplemental solution concurrently also applied, must be controlledduring such applications and properly directed to these detectors. Thiscan be accomplished by providing enclosed receiving reservoirs ahead ofthe general magnetic detector locations and enclosed accumulationreservoirs thereafter, as well as one or more enclosed testing flowpassageways or pools at the general magnetic detector locations, andenclosed channels between them as conduits for permitting the samplesolutions under selected pressures to reach such reservoirs, passagewaysand pools without risk of contamination from outside sources thereof.

[0092]FIGS. 25, 26 and 27 show side views in layer diagrams of some ofthe magnetic detectors described above provided below passageways, poolsor channels through which assay sample solutions can be provided toreach the vicinity of these detectors. FIG. 25 shows such an arrangementfor the magnetic detector shown in FIG. 11, FIG. 26 shows such anarrangement for the magnetic detector shown in FIG. 8, and FIG. 27 showssuch an arrangement for the magnetic detector shown in FIG. 9. Again, asin FIG. 9 versus FIG. 8, FIG. 27 is like FIG. 26 except that a Si₃N₄portion of the outer passivation layer is removed from selected areas orsome portion of that layer thereover is removed with molecular bindinglayer 12 afterward added there, and flux concentrators 19 and 20 areadded for amplifying externally applied fields, or a coil and fluxconcentrator can be added for guiding on-chip developed magnetic fields.

[0093] In each of FIGS. 25, 26 and 27 an aluminum nitride (AlN) etchstop, 42, is first provided over the exposed surfaces shown in thecorresponding previous detector drawings (FIGS. 11, 8 and 9) to athickness of 300 Å by sputter deposition. Thereafter, a 5 μm positivephotoresist layer is coated over etch stop 42 except in FIG. 27 where amuch thicker layer is added to cover flux concentrators 19 and 20perhaps as much as 20 μm depending on the thickness chosen for the fluxconcentrators. The photoresist is given a hard cure by heating througheither a convection flow or on a hot plate sufficiently to cause thepatterned resist to reach a temperature in excess of 200° C. Thereafter,a Si₃N₄ layer is sputter deposited on the photoresist to a thickness of2000 Å. Standard photoresist methods are used to provide a maskingpattern on this silicon nitride layer leaving openings where this layerand the BCB therebelow is to be removed by etching which is accomplishedby reactive ion etching (RIE). A cured, patterned photoresist layer, 43,(43′ in FIG. 27) results which serves as a dielectric, or electricalinsulating material, base on the magnetic detector chip surface for alid to be provided thereon to complete enclosing the desired reservoirs,passageways, pools and channels. In addition, a silicon nitride bondingmaterial layer, 44, results thereon as a basis for attaching such a lid.

[0094] The polymer material chosen for layer 43 or 43′ must be chosenwith some care. The temperature at which it can be hard cured, orcross-linked, must be low enough to avoid damaging the magneticdetectors. The resulting material in the layer should exhibit low waterabsorption, and should further exhibit sufficient mechanical stiffnessto support a lid and its attachment and to withstand the needed fluidpressures. The glass transition temperature of the material in layers 43or 43′ must be sufficiently high to exceed temperatures reached duringthermosonic bonding. One suitable photoresist for use in formingdielectric polymer layers 43 or 43′ is B-staged bisbenzocyclobutene(BCB) available from Dow Chemical Company in Midland, Mich. under thetrade name CYCLOTENE in both photodefinable and nonphotodefinableversions.

[0095] In order to seal the desired reservoirs, passageways, pools andchannels, polydimethy siloxane (PDMS; Sylgard 184, Dow corning, Mich.)was found suitable as a lid material due to its ability to be covalentlyattached to a number of substrates, its aptitude for conformal adhesionto a nonplanar substrate, and the ease of incorporating fluidinterconnects within the polymer. In its native state cross-linked PDMSis hydrophobic with an idealized surface terminated by methyl groups.Exposure to an oxygen plasma affords a hydrophilic surface dominated byhydroxyl groups. When brought into contact with a silanol terminatedsurface (e.g., glass) an irreversible bond is formed. The mechanism ofthis linkage is believed to occur through a condensation reactionbetween the SiOH groups of the oxygen plasma treated PDMS and those ofthe substrate (Duffy). Si₃N₄ was chosen for the bonding layer 44 becauseits surface possesses SiO₂ and SiOH groups and it is easily deposited atmicron dimensions on BCB.

[0096] A lid, 45, is constructed by curing PDMS at 70° C. on a positivemold that resulted in nanoliter chambers on the bonding surface of thecover. When the cover is aligned properly with layer 44, these chamberscoincide with the desired reservoirs. Layer 44 provided a surface thatformed an irreversible bond with the PDMS in lid 45 after oxygen plasmatreatment.

[0097] The oxygen plasma treatment of the PDMS and the Si₃N₄ isperformed in a series of steps. First, the integrated circuit chip withthe magnetic detectors and layer 44 is rinsed with isopropanol and driedunder flowing N₂. The chip is then treated for two minutes at an oxygenpressure of 800 mtorr and a forward power of 300 W in an RF generatedplasma. Both the PDMS and chip are then placed in the oxygen plasma fortwelve seconds, removed and placed in contact with each other. The chipassembly is then heated to 70° C. for ten minutes.

[0098] A top view of a portion of a more complete monolithic integratedcircuit chip magnetic detector based assay sample solution analysissystem is shown in FIG. 28 based on the interleaved magnetic fielddetector magnetoresistors of FIG. 14 but with theunder-the-magnetoresistor interconnection arrangement of FIGS. 11 and 25rather than the over-the-resistor interconnection arrangement of FIGS.8, 9, 26 and 27 actually shown in FIG. 14. Lid 45 is shown in place withan inlet reservoir, 50, provided by an inlet pool, 51, formed in the BCBbase and in the integrated circuit chip, and an inlet chamber, 52,molded into lid 45. An inlet capillary, 53, is inserted from the side ofPDMS material lid 45 therethrough into inlet chamber 52 that can beconnected to a syringe to supply the sample solution for analysis. Inletcapillary 53 is a polyimide coated fused silica capillaries (350 μm OD,250 μm ID; Polymicro Technologies, AZ).

[0099] Constant pressure applied to a connected syringe containing thesample solution will induce fluid flow through the capillary to inletreservoir 50 from where, after the filling of that reservoir, the fluidwill flow through an inlet channel, 54, to an analysis pool, 55, overmagnetoresistors 34 of the magnetic field detector bridge circuit. Afterthe detection of any magnetic fields associated with the samplesolution, the solution travels on to an outlet channel, 56, to an outletreservoir, 57, provided by an outlet pool, 58, formed in the BCB baseand in the integrated circuit chip, and an outlet chamber, 59, moldedinto lid 45. An outlet capillary, 60, is inserted from the side of PDMSmaterial lid 45 therethrough into outlet chamber 59 from which thesample solution can exit. Inlet pool 51, inlet channel 54, analysis pool55, outlet channel 56 and outlet pool 58 are shown in solid lines forclarity, as are flux concentrators 19 and 20, even though they are allpositioned below the top of lid 45.

[0100] A side section view in a layer diagram of a portion of what isshown in FIG. 28 as marked there is shown in FIG. 29. This view is takenfrom inlet reservoir 50 looking toward outlet reservoir 57 through inletchannel 54 and analysis pool 55. A closer look at analysis pool 55 isgiven in a top view thereof in FIG. 30 as marked in FIG. 29.

[0101] In operating a bridge circuit with the magnetic field detectorsbased on magnetoresistors as described above, the absence of anymagnetized beads over a magnetoresistor results in that magnetoresistoroperating on its basic resistance (R) or magnetoresistor output voltage(v, acquired from the resistance change bypassing a sense currenttherethrough) versus the externally applied magnetic field (H_(a))characteristic such as that shown by the solid line characteristic inFIG. 31. The resistance is relatively high near to zero fields where themagnetizations of the two ferromagnetic layers in such a magnetoresistorare directed away from one another, and decreases in the presence oflarger magnitude applied magnetic fields as the magnetizations of eachlayer are forced toward a common direction thereby.

[0102] The addition of magnetized beads very close to such amagnetoresistor does not alter the general shape of the resistanceversus applied magnetic field characteristic thereof but effectivelyspreads it wider over the applied magnetic field axis as those beads actto shunt part of the applied magnetic field away from thatmagnetoresistor. The result is shown as the dashed line resistance ormagnetoresistor output voltage versus the externally applied magneticfield characteristic in FIG. 31. The shunting effect shown here is about10% ofthe total field, i.e. it takes 10% more field to saturate the GMRresistor completely covered with beads than to saturate one with nobeads. Fewer beads near the magnetoresistor results in a smaller effect.

[0103] A bridge circuit magnetic field detection arrangement such asthat shown in FIG. 7 can be used to make accurate measurements of thesurface bead concentration near sensing magnetoresistors by having twoopposite bridge magnetoresistor exposed to the possibility of magnetizedbead coverage and the other two not so exposed. Again a constant voltagesource is used to supply electrical power to the bridge circuit and ahigh impedance amplifier is used to provide the bridge output signal avoltage measuring device as a basis for monitoring the bridge outputsignal. The bridge circuit magnetoresistors are assumed to be wellmatched to one another so that the characteristics of thosemagnetoresistors in the absence of magnetized beads are all like that ofthe solid line characteristic in FIG. 31, and like that of the dashedline characteristic in FIG. 31 for both of the pair of magnetoresistorsthat can be exposed to magnetized beads.

[0104] If the pair of bridge circuit resistors exposed to havingmagnetized beads nearby have the maximum number of such beads in theeffective sensing vicinity thereof present, seemingly a good referencemeasurement of the bridge output can be made at zero externally appliedmagnetic field because the resistances of all resistors will benominally equal (i.e., when the beads are not magnetized so that theyare not shunting away any of the magnetizing field from themagnetoresistors) for well matched magnetoresistors. Thereafter, singlepolarity or bipolar externally applied magnetic field value excursionsabout the zero field value can be applied, as indicated by thehorizontal double headed arrow crossing the zero applied field value inFIG. 31, to measure bridge circuit output signal voltages from thisreference to determine the number of beads present near the sensing pairof magnetoresistors. However, the resistances of magnetoresistors atzero applied magnetic field actually drift with time due to theconsiderable magnetic hysteresis in the GMR material used in theirconstruction as is indicated by the dashed lines in the characteristicsshown in FIG. 31 near zero externally applied magnetic fields. Thishysteresis leads to random magnetization orientation changes that appearas output signal noise and drift in the measurement.

[0105] A more stable null measurement can be made at larger externallyapplied magnetic fields. Above the externally applied magnetic fieldvalue H_(saturate) in FIG. 31, both pairs of bridge magnetoresistors aremagnetically saturated and so again have equal resistance values forwell matched magnetoresistors. Thus, the bridge circuit magnetoresistorresistances at H_(saturate) are balanced and the bridge output signalvalue is zero. At a lower externally applied magnetic field value ofH_(sense) below H_(saturate), the resistances of the two pairs of bridgemagnetoresistors are unequal due to the shunting effect of themagnetized beads being in the effective sensing vicinity of one of thosepairs. Thus, the bridge circuit magnetoresistor resistances at H_(sense)are now unbalanced and the bridge output signal becomes nonzero.

[0106] Hence, the number of magnetized beads present near the pair ofmagnetoresistors exposed thereto can be measured by providing anexternally applied field that changes from H_(saturate) to H_(sense) asindicated by the horizontal double headed arrow therebetween in FIG. 31.The effective output signals of the measurement system, then, are thedifferences between bridge circuit voltage outputs that occur at theexternally applied field value H_(saturate) (zero since there is nodifference the characteristics of the two pairs at that applied fieldvalue) and those that occur at the applied field value H_(sense) (whichis based on the difference between the solid line and dashed linecharacteristics at that applied field value) as indicated by thevertical double headed arrow showing the difference between the solidand dashed line characteristics at that applied field value.

[0107] This differential measurement technique (based on two differentexternally applied fields) also permits canceling of low frequency (1/f)noise otherwise resulting in individual measurements. A preferredarrangement would be to oscillate the externally applied field betweenH_(sense) and H_(saturate) at a frequency above the corner frequency ofthe 1/f noise (say 25 kHz) to thereby be operating where this noise is aminimum, and then measure the peak-to-peak value of the output. Typicalvalues for H_(sense) and H_(saturate) would be 300 Oe and 400 Oe,respectively, for multilayer material. Other GMR materials that saturatemore easily would have H_(sense) and H_(saturate) of 60 and 80 Oe. Thereare several possible types of GMR material, each with good and badfeatures. Those requiring high fields to saturate have good linearityand low hysteresis. Those requiring lower fields to saturate have morehysteresis and may require too much current density for the heat budgetof the measurement. Regardless of which GMR material is used, the keyrequirement is to make a reference measurement above the saturationfield of the resistors exposed to beads and a sense measurement at afield where both pairs are not saturated.

[0108] In a practical miniaturized system, it is not practical togenerate 300-400 Oe fields over any volume due to the electrical powerrequirements for doing so. One way to increase the effective field atthe measurement bridge is to use flux concentrators to “amplify” thefield. A typical amplification factor would be 15×. In this case, only20 to 27 Oe would be required. This is much easier to accomplish interms of the needed electrical power. Oscillating the externally appliedfield between H_(sense) and H_(saturate) at a selected frequency can beaccomplished in an arrangement like that shown in FIG. 10.

[0109] Although the present invention has been described with referenceto preferred embodiments, workers skilled in the art will recognize thatchanges may be made in form and detail without departing from the spiritand scope of the invention.

1. A method for operating a ferromagnetic thin-film based magnetic fielddetection system comprising a substrate, a plurality of magnetic fieldsensors capable of sensing externally applied magnetic fields up to asaturation magnitude value that are supported on said substrate of whichat least one can have magnetically permeable particles providedsufficiently close thereto to be capable of sensing resulting variationsin said externally applied magnetic fields and of which at least one issubstantially unaffected by such magnetically permeable particles, andan output circuit electrically connected to said plurality of magneticfield sensors to provide an indication of changes therefrom due tosensings of externally applied magnetic fields thereby, said methodcomprising: applying an external magnetic field primarily along a firstdirection to said plurality of magnetic field sensors of a magnitudesubstantially equaling or exceeding said saturation value; applying anexternal magnetic field primarily along said first direction to saidplurality of magnetic field sensors of a magnitude less than saidsaturation value; and measuring changes in said indications provided bysaid output circuit resulting in said applyings of said externalmagnetic fields.
 2. The method of claim 1 wherein said plurality ofmagnetic field sensors are connected in a bridge circuit.
 3. The methodof claim 1 wherein said applying of an external magnetic field primarilyalong a first direction to said plurality of magnetic field sensors of amagnitude substantially equaling or exceeding said saturation value andsaid applying of an external magnetic field primarily along said firstdirection to said plurality of magnetic field sensors of a magnitudeless than said saturation value is done alternately at a frequency atwhich 1/f noise has relatively small magnitudes.
 4. The method of claim3 wherein said frequency of alternation equals or exceeds 25 KHz.